EP4363572A1 - Procédé de synthèse de composés oligomères modifiés par liaison - Google Patents

Procédé de synthèse de composés oligomères modifiés par liaison

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Publication number
EP4363572A1
EP4363572A1 EP22834149.1A EP22834149A EP4363572A1 EP 4363572 A1 EP4363572 A1 EP 4363572A1 EP 22834149 A EP22834149 A EP 22834149A EP 4363572 A1 EP4363572 A1 EP 4363572A1
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EP
European Patent Office
Prior art keywords
certain embodiments
modified
sugar moiety
oligonucleotide
substituted
Prior art date
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EP22834149.1A
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German (de)
English (en)
Inventor
Andrew A. RODRIGUEZ
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Ionis Pharmaceuticals Inc
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Ionis Pharmaceuticals Inc
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Publication of EP4363572A1 publication Critical patent/EP4363572A1/fr
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    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111General methods applicable to biologically active non-coding nucleic acids
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    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00Processes for the preparation of sugar derivatives
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/314Phosphoramidates
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/315Phosphorothioates
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/32Chemical structure of the sugar
    • C12N2310/3212'-O-R Modification
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/33Chemical structure of the base
    • C12N2310/334Modified C
    • C12N2310/33415-Methylcytosine
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/35Nature of the modification
    • C12N2310/351Conjugate
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    • C12N2310/00Structure or type of the nucleic acid
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    • C12N2310/35Nature of the modification
    • C12N2310/352Nature of the modification linked to the nucleic acid via a carbon atom
    • C12N2310/3525MOE, methoxyethoxy
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    • C12N2310/00Structure or type of the nucleic acid
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    • C12N2310/35Nature of the modification
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    • C12N2330/00Production
    • C12N2330/30Production chemically synthesised

Definitions

  • the present disclosure provides stabilized reagent compositions for the synthesis of oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprising a modified oligonucleotide having at least one modified intemucleoside linking group.
  • antisense technology The principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates the amount, activity, and/or function of the target nucleic acid. For example, in certain instances, antisense compounds result in altered transcription or translation of a target. Such modulation of expression can be achieved by, for example, target RNA degradation or occupancy -based inhibition.
  • modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound.
  • RNAi refers to antisense-mediated gene silencing through a mechanism that utilizes the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • An additional example of modulation of RNA target function is by an occupancy -based mechanism such as is employed naturally by microRNA.
  • MicroRNAs are small non-coding RNAs that regulate the expression of proteincoding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. MicroRNA mimics can enhance native microRNA function. Certain antisense compounds alter splicing of pre-mRNA.
  • antisense compounds in a CRISPR system. Regardless of the specific mechanism, sequence-specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of disease.
  • Antisense technology is an effective means for modulating the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications.
  • Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, tolerability, pharmacokinetics, or affinity for a target nucleic acid.
  • Conjugate groups may be attached to an antisense compound to enhance one or more properties, such as pharmacokinetics, pharmacodynamics, and uptake into cells and/or tissues of interest.
  • Oligomeric compounds comprising an oligonucleotide are chemically synthesized in a multi-step process.
  • Substituted sulfonyl azides are useful reagents for synthesis of linkage modified oligonucleotides, but they can be high energy materials and dangerous to work with especially at the manufacturing scale.
  • Development of new stabilized reagent compositions and reaction conditions to ameliorate potentially dangerous chemistries on scale remains an important challenge.
  • oligomeric compounds including oligomeric compounds that are antisense agents or portions thereof
  • modified oligonucleotides consisting of linked nucleosides linked through intemucleoside linking groups, wherein at least one of the intemucleoside linking groups has Formula I:
  • the methods may comprise additing stabilizing materials to compositions of high energy reagents.
  • each SEQ ID NO contained herein is independent of any modification to a sugar moiety, an intemucleoside linkage, or a nucleobase.
  • compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an intemucleoside linkage, or a nucleobase.
  • sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications.
  • RNA or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary.
  • an oligonucleotide comprising a nucleoside comprising a 2’-OH(H) sugar moiety and a thymine base could be described as a DNA having a modified sugar (2’ -OH in place of one 2’-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) in place of an uracil of RNA).
  • nucleic acid sequences provided herein, including, but not limited to those in the sequence listing are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • a modified oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any modified oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and modified oligonucleotides having other modified nucleobases, such as “AT m CGAUCG,” wherein m C indicates a cytosine base comprising a methyl group at the 5-position.
  • furanosyl sugar moiety or nucleoside comprising a furanosyl sugar moiety means the furanosyl sugar moiety or nucleoside comprising the furanosyl sugar moiety comprises a substituent other than H or OH at the 2’-position and is a non-bicyclic furanosyl sugar moiety.
  • 2’ -substituted furanosyl sugar moieties do not comprise additional substituents at other positions of the furanosyl sugar moiety other than a nucleobase and/or intemucleoside linkage(s) when in the context of an oligonucleotide.
  • furanosyl sugar moiety or nucleoside comprising a furanosyl sugar moiety means the furanosyl sugar moiety or nucleoside comprising the furanosyl sugar moiety comprises a substituent other than H at the 4’-position and is a non-bicyclic furanosyl sugar moiety.
  • 4’ -substituted furanosyl sugar moieties do not comprise additional substituents at other positions of the furanosyl sugar moiety other than a nucleobase and/or intemucleoside linkage(s) when in the context of an oligonucleotide.
  • furanosyl sugar moiety or nucleoside comprising a furanosyl sugar moiety means the furanosyl sugar moiety or nucleoside comprising the furanosyl sugar moiety comprises a substituent other than H at the 5’-position and is a non-bicyclic furanosyl sugar moiety.
  • 5’ -substituted furanosyl sugar moieties do not comprise additional substituents at other positions of the furanosyl sugar moiety other than a nucleobase and/or intemucleoside linkage(s) when in the context of an oligonucleotide.
  • administering refers to routes of introducing a compound or composition provided herein to a subject to perform its intended function.
  • routes of administration include, but are not limited to, administration by inhalation, subcutaneous injection, intrathecal injection, and oral administration.
  • antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense oligonucleotide to its target nucleic acid.
  • antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense oligonucleotide.
  • antisense agent means an antisense oligonucleotide or an oligonucleotide duplex comprising an antisense oligonucleotide.
  • antisense compound means an antisense oligonucleotide or an oligonucleotide duplex comprising an antisense oligonucleotide.
  • antisense oligonucleotide means an oligonucleotide that is complementary to a target nucleic acid and is capable of achieving at least one antisense activity.
  • Antisense oligonucleotides include but are not limited to RNAi antisense modified oligonucleotides and RNase H antisense modified oligonucleotides.
  • an antisense oligonucleotide is paired with a sense oligonucleotide to form an oligonucleotide duplex.
  • an antisense oligonucleotide is unpaired and is a single-stranded antisense oligonucleotide.
  • an antisense oligonucleotide comprises a conjugate group.
  • artificial mRNA compound is a modified oligonucleotide, or portion thereof, having a nucleobase sequence comprising one or more codons.
  • bicyclic nucleoside or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • bicyclic sugar or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure.
  • the first ring of the bicyclic sugar moiety is a furanosyl moiety
  • the bicyclic sugar moiety is a modified bicyclic furanosyl sugar moiety.
  • the bicyclic sugar moiety does not comprise a furanosyl moiety.
  • capping reagent means a reagent effective to protect hydroxyl groups during oligonucleotide synthesis, e.g., synthesis on a solid support.
  • the capping reagent may be acetic anhydride.
  • a capping reagent may be delivered in a composition including a base and a solvent.
  • a capping reagent composition may include acetic anhydride and acetonitrile, or pyridine, N-methylimidazole (NMI), and acetonitrile.
  • cEt or “constrained ethyl” or “cEt sugar moiety” means a bicyclic sugar moiety, wherein the first ring of the bicyclic sugar moiety is a ribosyl sugar moiety, the second ring of the bicyclic sugar is formed via a bridge connecting the 4’ -carbon and the 2’ -carbon, the bridge has the formula 4'-CH(CH 3 )-O-2', and the methyl group of the bridge is in the S configuration.
  • a cEt bicyclic sugar moiety is in the ⁇ -D configuration.
  • oligonucleotide in reference to an oligonucleotide means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions.
  • Complementary nucleobases are nucleobase pairs that are capable of forming hydrogen bonds with one another.
  • Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methyl cytosine ( m C) and guanine (G).
  • Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. As used herein, “fully complementary” or “100% complementary” in reference to oligonucleotides means that such oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
  • conjugate group means a group of atoms consisting of a conjugate moiety and a conjugate linker.
  • conjugate moiety means a group of atoms that modifies one or more properties of a molecule compared to the identical molecule lacking the conjugate moiety, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • conjugate linker means a group of atoms comprising at least one bond.
  • CRISPR compound means a modified oligonucleotide that comprises a DNA recognition portion and a tracrRNA recognition portion.
  • DNA recognition portion is nucleobase sequence that is complementary to a DNA target.
  • tracrRNA recognition portion is a nucleobase sequence that is bound to or is capable of binding to tracrRNA. The tracrRNA recognition portion of crRNA may bind to tracrRNA via hybridization or covalent attachment.
  • cytotoxic or “cytotoxicity” in the context of an effect of an oligomeric compound or a parent oligomeric compound on cultured cells means an at least 2-fold increase in caspase activation following administration of 10 mM or less of the oligomeric compound or parent oligomeric compound to the cultured cells relative to cells cultured under the same conditions but that are not administered the oligomeric compound or parent oligomeric compound.
  • cytotoxicity is measured using a standard in vitro cytotoxicity assay.
  • deoxy region means a region of 5-12 contiguous nucleotides, wherein at least 70% of the nucleosides are stereo-standard DNA nucleosides.
  • each nucleoside is selected from a stereostandard DNA nucleoside (a nucleoside comprising a [l-D-2 -deoxy ribosyl sugar moiety), a stereo-non-standard nucleoside of Formula I- VII, a bicyclic nucleoside, and a substituted stereo-standard nucleoside.
  • a deoxy region supports RNase H activity.
  • a deoxy region is the gap of a gapmer.
  • double-stranded antisense compound means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an antisense oligonucleotide.
  • expression includes all the functions by which a gene’s coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation.
  • modulation of expression means any change in amount or activity of a product of transcription or translation of a gene. Such a change may be an increase or a reduction of any amount relative to the expression level prior to the modulation.
  • gapmer means an oligonucleotide having a central region comprising a plurality of nucleosides that support RNase H cleavage positioned between a 5 ’ -region and a 3 ’ -region.
  • the nucleosides of the 5’-region and 3’-region each comprise a 2 ’-substituted furanosyl sugar moiety or abicyclic sugar moiety
  • the 3’- and 5 ’-most nucleosides of the central region each comprise a sugar moiety independently selected from a 2’- deoxyfuranosyl sugar moiety or a sugar surrogate.
  • the positions of the central region refer to the order of the nucleosides of the central region and are counted starting from the 5’-end of the central region. Thus, the 5’-most nucleoside of the central region is at position 1 of the central region.
  • the “central region” may be referred to as a “gap”, and the “5’-region” and “3’-region” may be referred to as “wings”. Gaps of gapmers are deoxy regions.
  • hepatotoxic in the context of a mouse means a plasma ALT level that is above 300 units per liter. Hepatotoxicity of an oligomeric compound or parent oligomeric compound that is administered to a mouse is determined by measuring the plasma ALT level of the mouse 24 hours to 2 weeks following at least one dose of 1-150 mg/kg of the compound.
  • hepatotoxic in the context of a human means a plasma ALT level that is above 150 units per liter. Hepatotoxicity of an oligomeric compound or parent oligomeric compound that is administered to a human is determined by measuring the plasma ALT level of the human 24 hours to 2 weeks following at least one dose of 10-300 mg of the compound.
  • hybridization means the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • inhibiting the expression or activity refers to a reduction or blockade of the expression or activity relative to the expression or activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.
  • intemucleoside linkage or “intemucleoside linking group” means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • modified intemucleoside linkage means any intemucleoside linkage other than a naturally occurring, phosphodiester intemucleoside linkage.
  • Phosphorothioate linkage means a modified intemucleoside linkage in which one of the non-bridging oxygen atoms of a phosphodiester is replaced with a sulfur atom. Modified intemucleoside linkages may or may not contain a phosphoms atom.
  • a modified intemucleoside linkage may optionally comprise a conjugate group.
  • linked nucleosides are nucleosides that are connected in a continuous sequence ( i.e . no additional nucleosides are present between those that are linked).
  • maximum tolerated dose means the highest dose of a compound that does not cause unacceptable side effects.
  • the maximum tolerated dose is the highest dose of a modified oligonucleotide that does not cause an ALT elevation of three times the upper limit of normal as measured by a standard assay.
  • modulating refers to changing or adjusting a feature in a cell, tissue, organ or organism.
  • MOE means O-methoxy ethyl.
  • 2’-MOE” or“2’-O-methoxyethyl” means a2’-OCH 2 CH 2 OCH 3 group at the 2’ -position of a furanosyl ring.
  • the 2’-OCH 2 CH 2 OCH 3 group is in place of the 2’- OH group of a ribosyl ring or in place of a 2’-H in a 2’-deoxyribosyl ring.
  • a “2’-MOE sugar moiety” is a sugar moiety with a 2’-OCH 2 CH 2 OCH 3 group in place of the 2’-OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2’-MOE sugar moiety is in the ⁇ -D ribosyl configuration.
  • a “2’-OMe sugar moiety” is a sugar moiety with a 2’-CH 3 group in place of the 2’-OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2’-OMe sugar moiety is in the ⁇ -D ribosyl configuration and is a “stereo-standard 2’OMe sugar moiety”.
  • a “2’-F sugar moiety” is a sugar moiety with a 2’-F group in place of the 2’-OH group of a furanosyl sugar moiety. Unless otherwise indicated, a 2’-F sugar moiety is in the ⁇ -D ribosyl configuration and is a “stereo-standard 2’-F sugar moiety”.
  • motif means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or intemucleoside linkages, in an oligonucleotide.
  • nucleobase means an unmodified nucleobase or a modified nucleobase.
  • an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G).
  • a modified nucleobase is a group of atoms capable of pairing with at least one unmodified nucleobase.
  • a universal base is a nucleobase that can pair with any one of the five unmodified nucleobases.
  • 5-methylcytosine ( m C) is one example of a modified nucleobase.
  • nucleobase sequence means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar moiety or intemucleoside linkage modification.
  • nucleoside means a moiety comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified.
  • modified nucleoside means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. A modified nucleoside may comprise a conjugate group.
  • oligomeric compound means a compound consisting of (1) an oligonucleotide (a single- stranded oligomeric compound) or two oligonucleotides hybridized to one another (a double-stranded oligomeric compound); and (2) optionally one or more additional features, such as a conjugate group or terminal group which may be attached to the oligonucleotide of a single-stranded oligomeric compound or to one or both oligonucleotides of a double- stranded oligomeric compound.
  • oligonucleotide means a strand of linked nucleosides connected via intemucleoside linkages, wherein each nucleoside and intemucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 12-80 linked nucleosides, and optionally a conjugate group or terminal group.
  • modified oligonucleotide means an oligonucleotide, wherein at least one nucleoside (a modified nucleoside) or intemucleoside linkage (a modified intemucleoside linkage) is modified.
  • unmodified oligonucleotide means an oligonucleotide that does not comprise any nucleoside modifications or intemucleoside modifications. Oligonucleotides are oligomeric compounds and oligonucleotides may be incorporated into oligomeric compounds having additional features. An oligonucleotide or modified oligonucleotide may comprise a linker group that links it to a solid support. The linker may be as described in Ravikumar et al., Org. Process Res. Dev. 2008, 12, 3, 399-410.
  • oligonucleotide intermediate means a compound or portion thereof that arises during synthesis of an oligonucleotide and that will ultimately form a portion of such oligonucleotide. Oligonucleotide intermediates include, but are not limited to linked nucleosides, intemucleoside linkages, conjugate groups, and modifications described herein and precursors thereof. In certain embodiments, an oligonucleotide intermediate is a hydroxy group attached to a solid support. In certain embodiments, an oligonucleotide intermediate is a number of linked nucleosides attached to a solid support.
  • a pharmaceutical composition means a mixture of substances suitable for administering to a subject.
  • a pharmaceutical composition may comprise an antisense compound and an aqueous solution.
  • RNAi agent means an antisense agent that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid.
  • RNAi agents include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
  • RNAi agents may comprise conjugate groups and/or terminal groups.
  • an RNAi agent modulates the amount, activity, and/or splicing of a target nucleic acid.
  • the term RNAi agent excludes antisense agents that act through RNase H.
  • RNAi oligonucleotide means an RNAi antisense modified oligonucleotide or a RNAi sense modified oligonucleotide.
  • RNAi antisense modified oligonucleotide means an oligonucleotide comprising a region that is complementary to a target sequence, and which includes at least one chemical modification suitable for RNAi.
  • RNAi antisense oligomeric compound means a single-stranded oligomeric compound comprising a region that is complementary to a target sequence, and which includes at least one chemical modification suitable for RNAi.
  • RNAi sense modified oligonucleotide means an oligonucleotide comprising a region that is complementary to a region of an RNAi antisense modified oligonucleotide, and which is capable of forming a duplex with such RNAi antisense modified oligonucleotide.
  • RNAi sense oligomeric compound means a single-stranded oligomeric compound comprising a region that is complementary to a region of an RNAi antisense modified oligonucleotide and/or an RNAi antisense oligomeric compound, and which is capable of forming a duplex with such RNAi antisense modified oligonucleotide and/or RNAi antisense oligomeric compound.
  • a duplex formed by an RNAi antisense modified oligonucleotide and/or an RNAi antisense oligomeric compound with a RNAi sense modified oligonucleotide and/or an RNAi sense oligomeric compound is referred to as a double-stranded RNAi compound (dsRNAi) or a short interfering RNA (siRNA).
  • dsRNAi double-stranded RNAi compound
  • siRNA short interfering RNA
  • RNase H agent means an antisense agent that acts, at least in part, through RNase H to modulate a target nucleic acid and/or protein encoded by a target nucleic acid.
  • RNase H agents are single-stranded.
  • RNase H agents are double-stranded.
  • RNase H compounds may comprise conjugate groups and/or terminal groups.
  • an RNase H agent modulates the amount or activity of a target nucleic acid.
  • the term RNase H agent excludes antisense agents that act principally through RISC/Ago2.
  • RNase H antisense modified oligonucleotide means an oligonucleotide comprising a region that is complementary to a target sequence, and which includes at least one chemical modification suitable for RNase H- mediated nucleic acid reduction.
  • RNAi compound means an antisense compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid.
  • RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
  • an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid.
  • the term RNAi compound excludes antisense oligonucleotides that act through RNase H.
  • single-stranded in reference to an antisense compound means such a compound consisting of one oligomeric compound that is not paired with a second oligomeric compound to form a duplex.
  • Selfcomplementary in reference to an oligonucleotide means an oligonucleotide that at least partially hybridizes to itself.
  • a compound consisting of one oligomeric compound, wherein the oligonucleotide of the oligomeric compound is selfcomplementary, is a single-stranded compound.
  • a single-stranded antisense or oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex, in which case the compound would no longer be single-stranded.
  • stabilized phosphate group refers to a 5’-chemical moiety that results in stabilization of a 5’- phosphate moiety of the 5’ -terminal nucleoside of an oligonucleotide, relative to the stability of an unmodified 5’- phosphate of an unmodified nucleoside under biologic conditions.
  • stabilization of a 5 ’-phophate group includes but is not limited to resistance to removal by phosphatases.
  • Stabilized phosphate groups include, but are not limited to, 5’- vinyl phosphonates and 5’ -cyclopropyl phosphonate.
  • stabilizing agent refers to a substance that when present in a solution, including but not limited to a reaction mixture, reduces the risk of explosion.
  • a “standard oxidizing agent” refers to oxidizing agents well understood in the art of oligonucleotide synthesis to oxidize phosphorous intemucleoside linkages, including but not limited to basic solvents, mixtures of a basic solvent such as 3-picoline, pyridine, 2,6-lutidine with Iodine and water, mixtures of Iodine, NMI, a basic solvent, and water. Further examples and description of oxidation methods are described in WO2020236618A1, the disclosure of which is incorporated in its entirety herein.
  • a “standard sulfurizing agent” refers to reagents well understood in the art of oligonucleotide synthesis to sulfurize phosphorous intemucleoside linkages, including but not limited to phenacetyldilsulfide or xanthane hydride. Further examples and description of oxidation methods are described in WO2020236618A1, the disclosure of which is incorporated in its entirety herein.
  • stereo-standard nucleoside means a nucleoside comprising a non-bicyclic furanosyl sugar moiety having the configuration of naturally occurring DNA and RNA as shown below.
  • a “stereo-standard DNA nucleoside” is a nucleoside comprising a [l-D-2 ‘ -dcoxyribosyl sugar moiety.
  • a “stereo-standard RNA nucleoside” is a nucleoside comprising a ⁇ -D-ribosyl sugar moiety.
  • a “substituted stereo-standard nucleoside” is a stereo-standard nucleoside other than a stereo-standard DNA or stereo-standard RNA nucleoside.
  • Ri is a T- substiuent and R 2 -R 5 are each H.
  • the 2’-substituent is selected from OMe, F, OCH 2 CH 2 OCH 3 , O-alkyl, SMe, or NMA.
  • R 1 -R 4 are H and R 5 is a 5’ -substituent selected from methyl, allyl, or ethyl.
  • the heterocyclic base moiety Bx is selected from uracil, thymine, cytosine, 5-methyl cytosine, adenine or guanine. In certain embodiments, the heterocyclic base moiety Bx is other than uracil, thymine, cytosine, 5-methyl cytosine, adenine or guanine.
  • stereo-standard RNA nucleoside means a nucleoside comprising a non-bicyclic furanosyl sugar moiety having a configuration other than that of a stereo-standard sugar moiety.
  • a “stereo-non-standard nucleoside” is a 2'- ⁇ -L-dcoxyribosyl sugar moiety, 2'- ⁇ -D-deoxyribosyl sugar moiety, 2'- ⁇ -L- deoxyribosyl sugar moiety, a 2'- ⁇ -D-dcoxyxylosyl sugar moiety, a 2'- ⁇ -L-dcoxyxylosyl sugar moiety, a 2'- ⁇ -D- deoxyxylosyl sugar moiety, a 2'- ⁇ -L-deoxyxylosyl sugar moiety, a 2 ' - P uo ro - b - D -a rab i no sy 1 sugar moiety, a 2'-PiiqGq- ⁇ - D-xylosyl sugar moiety, a 2'-fluoro- ⁇ -D-ribosyl sugar moiety, a 2'-fluoro- ⁇
  • stereo-standard sugar moiety means the sugar moiety of a stereo-standard nucleoside.
  • stereo-non-standard sugar moiety means the sugar moiety of a stereo-non-standard nucleoside.
  • substituted stereo-non-standard nucleoside means a stereo -non-standard nucleoside comprising a substituent other than the substituent corresponding to natural RNA or DNA.
  • a substituted stereo-non-standard nucleoside is a a 2'-fluoro- ⁇ -D-arabinosyl sugar moiety, a 2'-fluoro- ⁇ -D-xylosyl sugar moiety, a 2'- fluoro- ⁇ -D-ribosyl sugar moiety, a 2'-fluoro- ⁇ -D-arabinosyl sugar moiety, a 2'-fluoro- ⁇ -D-xylosyl sugar moiety, a 2'- fluoro- ⁇ -L-ribosyl sugar moiety, a 2'-fluoro- ⁇ -L-xylosyl sugar moiety, a 2'-fluoro- ⁇ -L-arabinosyl sugar moiety,
  • sulfonyl oxidizing agent means an agent that can effect transformation of a phosphite triester to a phosphoramidate.
  • the sulfonyl oxidizing agent has a structure wherein R is as defined as for Formula I.
  • R is methyl and the sulfonyl oxidizing agent is methanesulfonyl azide (“MsN 3 ”).
  • sugar moiety means an unmodified sugar moiety or a modified sugar moiety.
  • unmodified sugar moiety means a ⁇ -D-ribosyl moiety, as found in naturally occurring RNA, or a ⁇ -D-2 , -deoxyribosyl sugar moiety as found in naturally occurring DNA.
  • modified sugar moiety or “modified sugar” means a sugar surrogate or a furanosyl sugar moiety other than a ⁇ -D-ribosyl or a ⁇ -D-2 , -deoxyribosyl.
  • Modified furanosyl sugar moieties may be modified or substituted at a certain position(s) of the sugar moiety, or unsubstituted, and they may or may not be stereo-non-standard sugar moieties.
  • Modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.
  • sugar surrogate means a modified sugar moiety that does not comprise a furanosyl or tetrahydrofuranyl ring (is not a “furanosyl sugar moiety”) and that can link a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide.
  • Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.
  • target nucleic acid means a nucleic acid that an oligomeric compound, such as an antisense compound, is designed to affect.
  • an oligomeric compound comprises an oligonucleotide having a nucleobase sequence that is complementary to more than one RNA, only one of which is the target RNA of the oligomeric compound.
  • the target RNA is an RNA present in the species to which an oligomeric compound is administered.
  • therapeutic index means a comparison of the amount of a compound that causes a therapeutic effect to the amount that causes toxicity.
  • Compounds having a high therapeutic index have strong efficacy and low toxicity.
  • increasing the therapeutic index of a compound increases the amount of the compound that can be safely administered.
  • Embodiment 1 A method comprising contacting a first oligonucleotide intermediate having a phosphite triester intemucleoside linkage with at least one stabilizing agent and with an oxidizing solution comprising a sulfonyl oxidizing agent to form a second oligonucleotide intermediate having an intemucleoside linking group of Formula XIV:
  • R is selected from aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C 1 -C 6 alkoxy, C 1 -C 20 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, substituted C 1 -C 20 alkyl, substituted C 1 -C 6 alkenyl substituted C 1 -C 6 alkynyl, and a conjugate group.
  • Embodiment 2 The method of embodiment 1, wherein the sulfonyl oxidizing agent is methanesulfonyl azide (MsN 3 ).
  • Embodiment 3 The method of any of embodiments 1 to 2, wherein oxidizing solution comprises the at least one stabilizing agent .
  • Embodiment 4 The method of any of embodiments 1 to 2, wherein the oxidizing solution does not comprise the at least one stabilizing agent.
  • Embodiment 5 The method of any of embodiments 1 to 4, wherein the oxidizing solution comprises a solvent selected from acetonitrile, toluene, dichloromethane, pyridine, N-methyl-2-pyrrolidone, and combinations thereof.
  • a solvent selected from acetonitrile, toluene, dichloromethane, pyridine, N-methyl-2-pyrrolidone, and combinations thereof.
  • Embodiment 6 The method of any of embodiments 1 to 5, wherein the at least one stabilizing agent is selected from naphthalene, sulfolane, and triphenylphosphate.
  • Embodiment 7 The method of any of embodiments 1 to 5, wherein the at least one stabilizing agent is triphenylphosphate (TPP).
  • TPP triphenylphosphate
  • Embodiment 8 The method of any of embodiments 1 to 7, wherein the at least one stabilizing agent is TPP.
  • Embodiment 9 The method of any of embodiments 1 to 7, wherein the at least one stabilizing agent is TPP.
  • Embodiment 10 The method of any of embodiments 1 to 9, wherein at least one stabilizing agent is a non-crosslinked polymer.
  • Embodiment 11 The method of embodiment 10, wherein the non-crosslinked polymer is polystyrene.
  • Embodiment 12 The method of any of embodiments 1 to 11, wherein a residue obtained by evaporating the solvent from the oxidizing solution and the at least one stabilizing agent has combustion energy less than 500 J.g -1 .
  • Embodiment 13 The method of any of embodiments 1 to 11, wherein residue obtained by evaporating the solvent from the oxidizing solution and the at least one stabilizing agent has combustion energy less than 300 J.g -1 .
  • Embodiment 14 A method of synthesizing a modified oligonucleotide comprising at least one intemucleoside linkage of Formula I:
  • X is selected from O or S
  • R is selected from aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C 1 -C 6 alkoxy, C 1 -C 20 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, substituted C 1 -C 20 alkyl, substituted C 1 -C 6 alkenyl substituted C 1 -C 6 alkynyl, and a conjugate group; wherein the method comprises steps of: a) providing a solid support having a blocked hydroxyl group attached thereto; b) adding a deblocking agent to the reaction to deblock the blocked hydroxyl group to provide a free hydroxyl group; c) adding a nucleoside to the reaction to couple at the free hydroxyl group, wherein the nucleosdide comprises a phosphoramidite group and a blocked hydroxyl group
  • a sulfonyl oxidizing agent and at least one stabilizing agent selected from TPP to produce a sulfonyl phosphoramidate intemucleoside linkage e) optionally treating the sufonyl phosphoramidate, phosphate triester, or thiophosphate triester linkages with a mixture of capping reagents to cap any unreacted free hydroxyl groups; f) iteratively repeating steps b) through e) a predetermined number of times to provide the modified oligonucleotide, provided that at least one iteration includes step (d) 3; g) treating the modified oligonucleotide with triethylamine in acetonitrile; and thereby synthesizing the modified oligonucleotide comprising at least one intemucleoside linkage of Formula I.
  • Embodiment 15 The method of embodiment 14, wherein sulfonyl oxidizing agent is in an oxidizing solution comprising methanesulfonyl azide and the at least one stabilizing agent.
  • Embodiment 16 is a diagrammatic representation of Embodiment 16:
  • Embodiment 17 The method of any of embodiments 15 to 16, wherein the at least one stabilizing agent is TPP.
  • Embodiment 18 The method of any of embodiment s 14 to 17, wherein a residue obtained by evaporation of the solvent from the solution comprising the sulfonyl oxidizing agent and the at least one stabilizing agent has combustion energy less than 500 J.g -1 .
  • Embodiment 19 The method of any of embodiments 14 to 17, wherein a residue obtained by evaporation of the solvent from the solution comprising the sulfonyl oxidizing agent and the at least one stabilizing agent has combustion energy less than 300 J.g -1 .
  • Embodiment 20 The method of any of embodiments 14 to 19, wherein X is O and R is methyl.
  • Embodiment 21 The method of any of embodiments 1 to 20, wherein the modified oligonucleotide comprises 12 to 25 linked nucleosides.
  • Embodiment 22 The method of any of embodiments 14 to 21, comprising treating the modified oligonucleotide with ammonium hydroxide to remove protecting groups and cleave the modified oligonucleotide from the solid support.
  • compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprising or consisting of oligonucleotides consisting of linked nucleosides and having at least one intemucleoside linking group of Formula I:
  • R is selected from aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C 1 -C 6 alkoxy, C 1 -C 20 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, substituted C 1 -C 20 alkyl, substituted C 1 -C 6 alkenyl substituted C 1 -C 6 alkynyl, and a conjugate group.
  • X is O and R is methyl
  • the intemucleoside linking group of Formula I is an intemucleoside linking group of Formula II below.
  • compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprising or consisting of oligonucleotides consisting of linked nucleosides and having at least one intemucleoside linking group of Formula II:
  • compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprising or consisting of oligonucleotides consisting of linked nucleosides and having at least one intemucleoside linking group of Formula III:
  • compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprising or consisting of oligonucleotides consisting of linked nucleosides and having at least one intemucleoside linking group of Formula IV:
  • Modified oligonucleotides comprise at least one modification relative to an unmodified oligonucleotide (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety, a stereo-non-standard nucleoside, and/or a modified nucleobase) and/or at least one modified intemucleoside linkage).
  • the modified intemucleoside linkage is a modified intemucleoside linking group having any of Formula I-IV.
  • compounds described herein are oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) having at least one modified intemucleoside linking group having any of Formula I-IV.
  • the present disclosure provides synthetic methods for preparing oligonucleotides comprising at least one modified intemucleoside linkage of the Formula I:
  • the present disclosure also provides synthetic methods for preparing oligomeric compounds comprising such oligonucleotides, where such oligomeric compounds comprise a conjugate moiety attached to the oligonucleotide through a cleavable linker.
  • the cleavable linker is a phosphate diester bond.
  • oligonucleotides having both at least one intemucleoside linkage of Formula I and at least one phosphorothioate diester linkage and/or at least one phosphate diester linkage have one or more desired properties.
  • oligonucleotides having at least one intemucleoside linkage of the Formula I are gapmers.
  • oligonucleotides having at least one intemucleoside linkage of the Formula I are used to modulate splicing of a nucleic acid target.
  • oligonucleotides having at least one intemucleoside linkage of the Formula I are RNAi compounds. Such RNAi compounds may be double-stranded or single-stranded. Such oligonucleotides may comprise any of the features, modified nucleosides, and nucleoside motifs described herein.
  • oligonucleotides may comprise any of the modified sugar moieties described herein and/or any of the modified nucleobases.
  • the synthetic processes described herein are used to synthesize oligomeric compounds comprising a conjugate group.
  • the synthetic processes described herein are used to synthesize oligomeric compounds comprising a conjugate group comprising one or more L- Acetylgalactosamine residues.
  • the oligomeric compounds synthesized using the processes described herein are gapmers.
  • oligomeric compounds synthesized using the processes described herein are RNAi compounds.
  • oligomeric compounds synthesized using the processes described herein are single-stranded.
  • oligomeric compounds synthesized using the processes described herein are double-stranded.
  • compounds synthesized using the processes described herein are formulated for administration to an animal.
  • the present disclosure provides certain sulfonyl oxidizing agents, for example, sulfonyl azides, for use in the synthesis of olignonucleotides comprising one or more sulfonyl phosphoramidate linkages.
  • the present disclosure further provides stabilizing agents that may be introduced to sulfonyl phosphoramidate linkage formation reactions.
  • the stabilizing agents may be introduced to the linkage formation reaction before, concomitantly with, or subsequent to introduction of the sulfonyl oxidizing agent, for example, sulfonyl azide.
  • more than one stabilizing agent may be introduced to the linkage formation reactions.
  • the stabilizing agents may ameliorate energetic events associated with use of sulfonyl azides.
  • the stabilizing agents may be sterically bulky compounds.
  • the stabilizing agents have low flammability properties.
  • the stabilizing agents have low volatility and may be solid or semi-solid at room temperature.
  • the sulfonyl azide and stabilizing agents may be dissolved in a solvent or solvent mixture prior to introduction to the linkage formation reaction.
  • Solvents that may be useful according to the present disclosure include, but are not limited to acetonitrile (MeCN), dichloromethane (DCM), toluene, pyridine, 7V-methyl-2- pyrrolidone (NMP), and mixtures thereof.
  • Solvents that may be useful according to the present disclosure include, but are not limited to acetonitrile (MeCN), toluene, dichloromethane (DCM), toluene, pyridine, Y- me thy 1 -2 - p v rro 1 i do nc (NMP), and mixtures thereof.
  • the solvents are acetonitrile and toluene.
  • the stabilizing agents may be solids or liquids at room temperature.
  • Stabilizing agents that are useful in the present disclosure include, but are not limited to naphthalene, sulfolane, and triphenylphosphate (TPP).
  • TPP triphenylphosphate
  • stabilizing agents for use in the present disclosure are non-crosslinked polymers, including but not limited to polystyrene. Additional stabilizing agents contemplated herein include soluble polymers, waxes, triglycerides, and paraffin wax.
  • a stabilizing agent should form a homogenous mixture with the sulfonyl oxidizing agent upon evaporation of a solvent. Stabilizing agents that are prone to crystallization are not believed to be suitable. Thus, diphenyl sulfone (DPS) was found to form crystals and is not suitable.
  • DPS diphenyl sulfone
  • a stabilizing agent described herein reduces the exotherm created during reaction of a sulfonyl oxidizing agent, such as methanesulfonyl azide.
  • the amount of stabilizing agent relative to the amount of sulfonyl oxidizing agent may be determined.
  • the amount of stabilizing agent provides a composition that can be handled and utilized in synthesis safely.
  • the stabilizing agent may be in an amount that permits synthesis of oligonucleotides on process scale, for example, oligonucleotides may be synthesized in amounts sufficient to carry out clinical trials.
  • the stabilizing agent may be easily removable by solvent or aqueous wash.
  • the stabilizing agent as used in synthesis of therapeutic oligonucleotides, may be compatible with GMP protocols.
  • the stabilizing agent provides a composition that does not have a risk of explosion upon impact, for example, when methanesulfonyl azide is the sulfonyl oxidizing agent.
  • a stabilized composition comprising methanesulfonyl azide and a stabilizing agent.
  • a stabilized composition comprising, consisting essentially, or consisting of sulfolane and methansulfonyl azide.
  • the stabilized composition may further comprise a solvent.
  • a stabilized composition comprising, consisting essentially, or consisting of sulfolane, methansulfonyl azide, and optionally acetonitrile.
  • the stabilized composition may optionally be placed in contact with a solid support carrying an oligonucleotide intermediate for use in the synthesis of oligomeric compounds that contain phosphoramidate intemucleoside linkages, as described herein.
  • processes described herein are useful for synthesizing oligomeric compounds comprising or consisting of oligonucleotides consisting of linked nucleosides.
  • the present disclosure provides reagents for use in the synthesis of oligonucleotides having any number of modifications described herein.
  • a stabilized composition comprising methanesulfonyl azide and sulfolane.
  • the composition may comprise 0.1 to 100 equivalents, 0.1 to 10 equivalents, 1 to 10 equivalents, 3 to 6 equivalents, 4 to 5 equivalents, 1 to 2 equivalents, 2 to 3 equivalents, 3 to 4 equivalents, 5 to 6 equivalents, 6 to 7 equivalents, 7 to 8 equivalents, 8 to 9 equivalents, or 9 to 10 equivalents of sulfolane relative to methanesulfonyl azide, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 equivalents of sulfolane relative to methanesulfonyl azide.
  • the concentration of methanesulfonyl azide in the stabilized composition may be 0.1 to 10 M, for example 0.5 to 10 M, 0.5 to 5 M, 0.1 to 5 M, 0.3 to 1.5 M, 0.4 to 0.6 M, 0.1 to 0.2 M, 0.2 to 0.3 M, 0.3 to 0.4 M, 0.4 to 0.5 M, 0.4 to 0.6 M, 0.5 to 0.6 M, 0.6 to 0.7 M, 0.7 to 0.8 M, 0.8 to 0.9 M, 0.9 to 1 M, 1 to 1.1 M, 1 to 1.2 M, 1.1 to 1.2 M, 1.1 to 1.3 M, 1.2 to 1.3 M, 1 to 1.5 M, 1.3 to 1.4 M, 1.4 to 1.5 M, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 M.
  • the concentration of sulfolane in the stabilized composition may be 0.1 to 20 M, for example 1 to 20 M, 1 to 10 M, 3 to 6 M, 4 to 5 M, 1 to 2 M, 2 to 3 M, 3 to 4 M, 5 to 6 M, 6 to 7 M, 7 to 8 M, 8 to 9 M, or 9 to 10 M, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 M.
  • Modified nucleosides comprise a stereo-non-standard nucleoside, or a modified sugar moiety, or a modified nucleobase, or any combination thereof.
  • modified sugar moieties are stereo-non-standard sugar moieties.
  • sugar moieties are substituted furanosyl stereo-standard sugar moieties.
  • modified sugar moieties are bicyclic or tricyclic furanosyl sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
  • modified sugar moieties are stereo-non-standard sugar moieties shown in Formulas V-XI below: one of Ji and J 2 is H and the other of Ji and J 2 is selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 , O- C 1 -C 6 alkoxy, and SCH 3 ; one of J 3 and J 4 is H and the other of J 3 and J 4 is selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 , O-C 1 -C 6 alkoxy, and SCH 3 ; and wherein one of J 5 and J 6 , is H and the other of J 5 and J 6 , is selected from H, OH, F, OCH 3 , OCH 2 CH 2 OCH 3 , O-C 1 -C 6 alkoxy, and SCH 3 ; and wherein one of J 7 and J 8 is H and the other of J 7 and J 8 is selected from H, OH, F, OCH 3 ,
  • modified sugar moieties are substituted stereo-standard furanosyl sugar moieties comprising one or more acyclic substituent, including but not limited to substituents at the 2’, 3’, 4’, and/or 5’ positions.
  • the furanosyl sugar moiety is a ribosyl sugar moiety.
  • one or more acyclic substituent of substituted stereo-standard sugar moieties is branched.
  • 2’ -substituent groups suitable for substituted stereo-standard sugar moieties include but are not limited to: 2’-F, 2'-OCH 3 (“2’-OMe” or “2’-O-methyl”), and 2'-O(CH 2 ) 2 OCH 3 (“2’-MOE”).
  • 2’-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3 , O-C 1 -C 10 alkoxy, O-C 1 -C 10 substituted alkoxy, C 1 -C 10 alkyl, C 1 -C 10 substituted alkyl, S-alkyl, N(R m )-alkyl, O-alkenyl, S-alkenyl, N(R m )-alkenyl, O-alkynyl, S-alkynyl, N(R m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 ON(R m )(R n ) or OCH 2 C
  • these 2'-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Examples of 3’-substituent groups include 3’-methyl (see Frier, et al., The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically -modified DNA:RNA duplexes. Nucleic Acids Res., 25, 4429-4443, 1997.)
  • Examples of 4’ -substituent groups suitable for substituted stereostandard sugar moieties include but are not limited to alkoxy (e.g. , methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128.
  • non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2'-F-5'-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.
  • 2’,4’-difluoro modified sugar moieties have been described in Martinez-Montero, et al., Rigid 2', 4'- difluororibonucleosides: synthesis, conformational analysis, and incorporation into nascent RNA by HCV polymerase. J. Org. Chem., 2014, 79:5627-5635.
  • Modified sugar moieties comprising a 2’ -modification (OMe or F) and a 4’- modification (OMe orF) have also been described in Malek-Adamian, et al., J. Org. Chem , 2018, 83: 9839-9849.
  • a 2’ -substituted stereo-standard nucleoside comprises a sugar moiety comprising a T- substituent group selected from: F, OCH 3 , and OCH 2 CH 2 OCH 3 .
  • the 4’ O of 2’-deoxyribose can be substituted with a S to generate 4’-thio DNA (see Takahashi, et al., Nucleic Acids Research 2009, 37: 1353-1362). This modification can be combined with other modifications detailed herein.
  • the sugar moiety is further modified at the 2’ position.
  • the sugar moiety comprises a 2’-fluoro. A thymidine with this sugar moiety has been described in Watts, ct al.. ./. Org. Chem. 2006, 71(3): 921-925 (4’-S-fluoro5-methylarauridine orFAMU).
  • nucleosides comprise modifed sugar moieties that comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a 4’ to 2’ bridge between the 4' and the 2' furanose ring atoms.
  • the furanose ring is a ribose ring.
  • each R, R a , and R b is, independently, H, a protecting group, or Ci-Ci 2 alkyl (see, e.g. Imanishi et al., U.S.
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • an LNA nucleoside (described herein) may be in the a-L configuration or in the ⁇ -D configuration.
  • general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA) are identified in exemplified embodiments herein, they are in the ⁇ -D configuration, unless otherwise specified.
  • modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5’ -substituted and 4’-2’ bridged sugars).
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom.
  • such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein.
  • certain sugar surrogates comprise a 4’-sulfur atom and a substitution at the 2'-position (see, e.g., Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and/or the 5’ position.
  • sugar surrogates comprise rings having other than 5 atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran (“THP”).
  • TTP tetrahydropyrans
  • Such tetrahydropyrans may be further modified or substituted.
  • Nucleosides comprising such modified tetrahydropyrans include but are not limited to hexitol nucleic acid (“HNA”), altritol nucleic acid (“ANA”), mannitol nucleic acid (“MNA”) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem.
  • F-HNA fluoro HNA
  • U.S. 8,088,904 see e.g.Swayze et al., U.S. 8,088,904; Swayze et al., U.S. 8,440,803; Swayze et al., U.S. 8,796,437; and Swayze et al., U.S. 9,005,906;
  • F-HNA can also be referred to as a F- THP or 3 '-fluoro tetrahydropyran).
  • sugar surrogates comprise rings having no heteroatoms.
  • nucleosides comprising bicyclo [3.1.0] -hexane have been described (see, e.g., Marquez, et al., J. Med. Chem. 1996, 39:3739-3749).
  • sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
  • nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5,698,685; Summerton et al., U.S. 5,166,315; Summerton et al., U.S. 5,185,444; and Summerton et al., U.S. 5,034,506).
  • morpholino means a sugar surrogate comprising the following structure:
  • morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure.
  • Such sugar surrogates are refered to herein as “modifed morpholinos.”
  • morpholino residues replace a full nucleotide, including the intemucleoside linkage, and have the structures shown below, wherein Bx is a heterocyclic base moiety.
  • sugar surrogates comprise acyclic moieites.
  • nucleosides and oligonucleotides comprising such acyclic sugar surrogates include but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar etal., Org. Biomol. Chem., 2013, 77, 5853-5865), glycol nucleic acid(“GNA”, see Schlegel, et al., J. Am. Chem. Soc. 2017, 139:8537-8546) and nucleosides and oligonucleotides described in Manoharan et al., WO2011/133876.
  • acyclic sugar surrogates are selected from:
  • modified sugar moieties comprise a conjugate group and/or a terminal group.
  • Modified sugar moieties are linked to conjugate groups through a conjugate linker.
  • modified furanosyl sugar moieties comprise conjugate groups attached at the 2’, 3’, or 5’ positions.
  • the 3’ -most sugar moiety of the nucleoside is modified with a conjugate group or a terminal group.
  • the 5’ -most sugar moiety of the nucleoside is modified with a conjugate group or a terminal group.
  • a sugar moiety near the 3 ’ end of the nucleoside is modified with a conjugate group.
  • a sugar moiety near the 5’ end of the nucleoside is modified with a conjugate group.
  • terminal groups include but are not limited to conjugate groups, capping groups, phosphate group, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.
  • terminal groups at the 5’ -terminus comprise a stabilized phosphate group.
  • the phosphorus atom of the stabilized phosphate group is attached to the 5’ -terminal nucleoside through a phosphorus-carbon bond.
  • the carbon of that phosphorus -carbon bond is in turn bound to the 5’-position of the nucleoside.
  • the oligonucleotide comprises a 5’ -stabilized phosphate group having the following formula: wherein:
  • R a and R c are each, independently, OH, SH, C 1 -C 6 , alkyl, substituted C 1 -C 6 , alkyl, C 1 -C 6 , alkoxy, substituted Ci- C 6 alkoxy, amino or substituted amino;
  • R b is O or S
  • X is substituted or unsubstituted C; and wherein X is attached to the 5’-terminal nucleoside.
  • X is bound to an atom at the 5’-position of the 5’-terminal nucleoside.
  • the 5’-atom is a carbon and the bond between X and the 5’-carbon of the 5’-terminal nucleoside is a carbon-carbon single bond. In certain embodiments, it is a carbon-carbon double bond. In certain embodiments, it is a carbon-carbon triple bond.
  • the 5’ -carbon is substituted.
  • X is substituted. In certain embodiments, X is unsubstituted.
  • the oligonucleotide comprises a 5’ -stabilized phosphate group having the following formula: wherein:
  • R a and R c are each, independently, OH, SH, C 1 -C 6 , alkyl, substituted C 1 -C 6 , alkyl, C 1 -C 6 , alkoxy, substituted C 1 -C 6 , alkoxy, amino or substituted amino;
  • R b is O or S
  • X is substituted or unsubstituted C
  • Y is selected from C, S, and N. In certain embodiments, Y is substituted or unsubstituted C.
  • the bond between X and Y may be a single-, double-, or triple-bond.
  • the stabilized phosphate group is 5’-vinyl phosphonate or 5’ -cyclopropyl phosphonate.
  • a terminal group at the 5’ -terminus is a 5’-mesyl phosphoramidate, having formula
  • a terminal group at the 5’ -terminus is a 5’-mesyl phosphoramidate, having formula
  • modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines.
  • modified nucleobases are selected from: 2-aminopropyladenine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-CoC-CH 3 ) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5- ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7
  • nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, 1,3- diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3-diazaphenoxazine-2-one (G-clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in Merigan et al., U.S.
  • modified nucleosides comprise double-headed nucleosides having two nucleobases. Such compounds are described in detail in Sorinas et al., J. Org. Chem, 201479: 8020-8030.
  • compounds comprise or consist of a modified oligonucleotide complementary to a target nucleic acid comprising one or more modified nucleobases.
  • the modified nucleobase is 5-methylcytosine.
  • each cytosine is a 5-methylcytosine.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I are selected over compounds lacking such intemucleoside linkages having Formula I because of one or more desirable properties.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced cellular uptake.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced affinity for target nucleic acids.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have increased stability in the presence of nucleases. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced bioavailability.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced RNase H activity. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced RNAi activity. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have enhanced CRISPR activity.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have reduced interactions with certain proteins. In certain embodiments, antisense agents, oligomeric compounds, and modified oligonucleotides described herein having one or more modified intemucleoside linkages having Formula I have increased interactions with certain proteins. Methods of making oligonucleotides having at least one intemucleoside linkage of Fommula I (including but not limited to Fomula II-IV) may be used to make oligomeric compounds having any of the above properties.
  • Fommula I including but not limited to Fomula II-IV
  • oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) comprise or consist of a modified oligonucleotide complementary to a target nucleic acid comprising one or more modified intemucleoside linkages having Formula I:
  • X is selected from O or S
  • R is selected from aryl, a substituted aryl, a heterocycle, a substituted heterocycle, an aromatic heterocycle, a substituted aromatic heterocycle, a diazole, a substituted diazole, a C 1 -C 6 alkoxy, C 1 -C 20 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, substituted C 1 -C 20 alkyl, substituted C 1 -C 6 alkenyl substituted C 1 -C 6 alkynyl, and a conjugate group.
  • antisense agents, oligomeric compounds, and modified oligonucleotides comprise one or more intemucleoside linkages of Formula I and one or more intemucleoside linkages that are not of Formula I.
  • such intemucleoside linkages are phosphorothioate linkages.
  • each intemucleoside linkage of an oligomeric compound other than the at least one intemucleoside linkage of Formula I is a phosphorothioate intemucleoside linkage.
  • each intemucleoside linkage of an oligomeric compound other than the at least one intemucleoside linkage of Formula I is a phosphorothioate intemucleoside linkage or a phosphodie intemucleoside linkage.
  • Phosphodiester intemucleoside linking group Phosphorothioate intemucleoside linking group
  • nucleosides of modified oligonucleotides may be linked together using any intemucleoside linkage.
  • the two main classes of intemucleoside linkages are defined by the presence or absence of a phosphoms atom.
  • Modified intemucleoside linkages compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non-phosphorous-containing intemucleoside linkages are well known to those skilled in the art.
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts. b. Chiral Intemucleoside Linkages
  • Representative intemucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates.
  • Modified oligonucleotides comprising intemucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom intemucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations.
  • populations of modified oligonucleotides comprise phosphorothioate intemucleoside linkages wherein all of the phosphorothioate intemucleoside linkages are stereorandom.
  • modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. All phosphorothioate linkages described herein are stereorandom unless otherwise specified. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate intemucleoside linkages in a particular, independently selected stereochemical configuration.
  • the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population.
  • modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017/015555.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (S'p) configuration.
  • a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration.
  • modified oligonucleotides comprising (Rp) and/or (S'p) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:
  • chiral intemucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.
  • an intemucleoside linkage of Formula I may comprise a chiral center.
  • modified oligonucleotides comprise chiral linkages of Formula II, as shown below. c. Alternatives to 5’ to 3’ Intemucleoside Linkages
  • nucleic acids can be linked 2 ’ to 5 ’ rather than the standard 3 ’ to 5 ’ linkage . Such a linkage is illustrated below.
  • nucleosides can be linked by 2’, 3 ’-phosphodiester bonds.
  • the nucleosides are threofuranosyl nucleosides (TNA; see Bala, et al., J Org. Chem. 2017, 82:5910-5916).
  • TNA threose nucleic acid
  • Additional modified linkages include ⁇ , ⁇ -D-CN A type linkages and related confonnationally -constrained linkages, shown below. Synthesis of such molecules has been described previously (see Dupouy, et al., Angew. Chem. Ini. Ed. Engl, 2014, 45: 3623-3627; Borsting, et al. Tetrahedron, 2004, 60:10955-10966; Ostergaard, et al., ACS Chem. Biol. 2014, 9: 1975-1979; Dupouy, etal., Eur. J Org.
  • an intemucleoside linking group may comprise a conjugate group.
  • an intemucleoside linking group of Formula I comprises a conjugate group.
  • the conjugate group of a modified oligonucleotide may be attached to the remainder of the modified oligonucleotide through a modified intemucleoside having Formula I:
  • R comprises a conjugate group.
  • the conjugate group comprises a cell-targeting moiety.
  • the conjugate group comprises a carbohydrate or carbohydrate cluster.
  • the conjugate group comprises N-acetylgalactosamine (GalNAc).
  • the conjugate group comprises a lipid.
  • the conjugate group comprises C 10 -C 20 alkyl.
  • the conjugate group comprises C 16 alkyl.
  • the intemucleoside linking group comprising a conjugate group has Formula IV:
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of oligonucleotides.
  • Modified oligonucleotides can be described by their motif, e.g. a pattern of unmodified and/or modified sugar moieties, nucleobases, and/or intemucleoside linkages.
  • modified oligonucleotides comprise one or more stereo-non-standard nucleosides.
  • modified oligonucleotides comprise one or more stereo-standard nucleosides.
  • modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar.
  • modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase.
  • modified oligonucleotides comprise one or more modified intemucleoside linkage.
  • the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleoside linkages of a modified oligonucleotide define a pattern or motif.
  • the patterns or motifs of sugar moieties, nucleobases, and intemucleoside linkages are each independent of one another.
  • a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or intemucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of oligonucleotides.
  • oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include without limitation any of the sugar modifications discussed herein.
  • a modified oligonucleotide comprises or consists of a gapmer.
  • the sugar motif of a gapmer defines the regions of the gapmer: 5’-region, central region (gap), and 3’-region.
  • the central region is linked directly to the 5’ -region and to the 3’ -region with no nucleosides intervening.
  • the central region is a deoxy region.
  • the nucleoside at the first position (position 1) from the 5’-end of the central region and the nucleoside at the last position of the central region are adjacent to the 5’-region and 3’-region, respectively, and each comprise a sugar moiety independently selected from a 2’-deoxyluranosyl sugar moiety or a sugar surrogate.
  • the nucleoside at position 1 of the central region and the nucleoside at the last position of the central region are DNA nucleosides, selected from stereo-standard DNA nucleosides or stereo-non-standard DNA nucleosides having any of formulas I- VII, wherein each J is H.
  • the nucleoside at the first and last positions of the central region adjacent to the 5’ and 3’ regions are stereo-standard DNA nucleosides.
  • the nucleosides at the other positions within the central region may comprise a 2’- substituted furanosyl sugar moiety or a substituted stereo-non-standard sugar moiety or a bicyclic sugar moiety.
  • each nucleoside within the central region supports RNase H cleavage.
  • a plurality of nucleosides within the central region support RNase H cleavage.
  • the lengths (number of nucleosides) of the three regions of a gapmer may be provided using the notation [# of nucleosides in the 5’ -region] - [# of nucleosides in the central region] - [# of nucleosides in the 3’- region].
  • a 3-10-3 gapmer consists of 3 linked nucleosides in each of the 3’ and 5’ regions and 10 linked nucleosides in the central region. Where such nomenclature is followed by a specific modification, that modification is the modification of each sugar moiety of each 5’ and 3’ -region and the central region nucleosides comprise stereostandard DNA sugar moieties.
  • a 5-10-5 MOE gapmer consists of 5 linked nucleosides each comprising 2’-MOE- stereo-standard sugar moieties in the 5’-region, 10 linked nucleosides each comprising a stereo-standard DNA sugar moiety in the central region, and 5 linked nucleosides each comprising 2’-MOE-stereo-standard sugar moieties in the 3’- region.
  • a 5-10-5 MOE gapmer having a substituted stereo-non-standard nucleoside at position 2 of the gap has a gap of 10 nucleosides wherein the 2 nd nucleoside of the gap is a substituted stereo-non-standard nucleoside rather than the stereo-standard DNA nucleoside.
  • Such oligonucleotide may also be described as a 5-1-1-8-5 MOE/substituted stereo- non-standard/MOE gapmer.
  • a 3-10-3 cEt gapmer consists of 3 linked nucleosides each comprising a cEt in the 5’- region, 10 linked nucleosides each comprising a stereo-standard DNA sugar moiety in the central region, and 3 linked nucleosides each comprising a cEt in the 3 ’-region.
  • a 3-10-3 cEt gapmer having a substituted stereo-non-standard nucleoside at position 2 of the gap has a gap of 10 nucleoside wherein the 2 nd nucleoside of the gap is a substituted stereo-non-standard nucleoside rather than the stereo-standard DNA nucleoside.
  • Such oligonucleotide may also be described as a 3-1-1-8-3 cEt/substituted stereo-non-standard/cEt gapmer.
  • the sugar motif of a 3-10-3 cEt gapmer may also be denoted by the notation kkk-d(10)-kkk, wherein each “k” represents a cEt and each “d” represents a 2 ' - ⁇ -D-dcoxyribosyl sugar moiety.
  • This sugar motif is independent of the nucleobase sequence, the intemucleoside linkage motif, and any nucleobase modifications.
  • a 5-10-5 MOE gapmer may be denoted by the notation eeeee-d(10)-eeeee or e(5)-d(10)-e(5), wherein each “e” represents a 2 ' -MOE- ⁇ -D- ribofuranosyl sugar moiety, and each “d” represents a 2 ' - ⁇ -D-dcoxyribosyl sugar moiety.
  • each nucleoside of a modified oligonucleotide, or portion thereof comprises a 2’- substituted sugar moiety, a bicyclic sugar moiety, a sugar surrogate, or a 2’-deoxyribosyl sugar moiety.
  • the 2’-substituted sugar moiety is selected from a 2’-MOE sugar moiety, a 2’-NMA sugar moiety, a 2’- OMe sugar moiety, and a 2’-F sugar moiety.
  • the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety.
  • the sugar surrogate is selected from morpholino, modified morpholino, PNA, THP, and F-HNA.
  • modified oligonucleotides comprise at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleosides comprising a modified sugar moiety.
  • the modified sugar moiety is selected independently from a 2 ’-substituted sugar moiety, a bicyclic sugar moiety, or a sugar surrogate.
  • the 2’-substituted sugar moiety is selected from a 2’-MOE sugar moiety, a 2’-NMA sugar moiety, a 2’-OMe sugar moiety, and a 2’-F sugar moiety.
  • the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety.
  • the sugar surrogate is selected from morpholino, modified morpholino, THP, and F-HNA.
  • each nucleoside of a modified oligonucleotide comprises a modified sugar moiety (“fully modified oligonucleotide”) ⁇
  • each nucleoside of a fully modified oligonucleotide comprises a 2’-substituted sugar moiety, abicyclic sugar moiety, or a sugar surrogate.
  • the 2’- substituted sugar moiety is selected from a 2’-MOE sugar moiety, a 2’-NMA sugar moiety, a 2’-OMe sugar moiety, and a 2’-F sugar moiety.
  • the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety.
  • the sugar surrogate is selected from morpholino, modified morpholino, THP, and F-HNA.
  • each nucleoside of a fully modified oligonucleotide comprises the same modified sugar moiety (“uniformly modified sugar motif’).
  • the uniformly modified sugar motif is 7 to 20 nucleosides in length.
  • each nucleoside of the uniformly modified sugar motif comprises a 2’-substituted sugar moiety, abicyclic sugar moiety, or a sugar surrogate.
  • the 2’-substituted sugar moiety is selected from a 2’-MOE sugar moiety, a 2’-NMA sugar moiety, a 2’-OMe sugar moiety, and a 2’-F sugar moiety.
  • the bicyclic sugar moiety is selected from a cEt sugar moiety and an LNA sugar moiety.
  • the sugar surrogate is selected from morpholino, modified morpholino, THP, and F- HNA.
  • modified oligonucleotides having at least one fully modified sugar motif may also comprise at least 1, at least 2, at least 3, or at least 42’-deoxyribonucleosides.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of oligonucleotides.
  • oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • each nucleobase is modified.
  • none of the nucleobases are modified.
  • each purine or each pyrimidine is modified.
  • each adenine is modified.
  • each guanine is modified.
  • each thymine is modified.
  • each uracil is modified.
  • each cytosine is modified.
  • some or all of the cytosine nucleobases in a modified oligonucleotide are 5-methylcytosines.
  • modified oligonucleotides comprise a block of modified nucleobases.
  • the block is at the 3 ’-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 3’-end of the oligonucleotide. In certain embodiments, the block is at the 5’-end of the oligonucleotide. In certain embodiments the block is within 3 nucleosides of the 5 ’-end of the oligonucleotide.
  • one nucleoside comprising a modified nucleobase is in the central region of a modified oligonucleotide.
  • the sugar moiety of said nucleoside is a 2 ‘ - ⁇ -D-dco. ⁇ yribosyl moiety.
  • the modified nucleobase is selected from: 5-methyl cytosine, 2-thiopyrimidine, 2- thiothymine, 6-methyladenine, inosine, pseudouracil, or 5-propynepyrimidine.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of oligonucleotides.
  • oligonucleotides comprise modified and/or unmodified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • the one or two 5 ’-most intemucleoside linkages are intemucleoside linkages of Formula I.
  • the one or two 3 ’-most intemucleoside linkages are intemucleoside linkages of Formula I.
  • each intemucleoside linkage is selected from an intemucleoside linkage of Formula I, a phosphorothioate intemucleoside linkage, and a phosphodiester intemucleoside linkage.
  • each intemucleoside linkage is selected from an intemucleoside linkage of Formula I and a phosphodiester intemucleoside linkage.
  • each phosphorothioate intemucleoside linkage is independently selected from a stereorandom phosphorothioate, a (Sp) phosphorothioate, and a (Rp) phosphorothioate.
  • the intemucleoside linkages within the central region of a modified oligonucleotide are all modified.
  • all of the phosphorothioate linkages are stereorandom.
  • all of the phosphorothioate linkages in the 5’ -region and 3’ -region are (Sp) phosphorothioates, and the central region comprises at least one .S'p. .S'p. //p motif.
  • populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such intemucleoside linkage motifs.
  • a double-stranded antisense compound is a double-stranded RNAi compound comprising an RNAi antisense modified oligonucleotide and an RNAi sense modified oligonucleotide, wherein one or both of the RNAi antisense modified oligonucleotide and/or RNAi sense oligomeric compound have one or more modified intemucleoside linking groups having Formula I.
  • the RNAi antisense modified oligonucleotide comprises at least two, at least three, at least four, at least five, or at least six modified intemucleoside linking groups having Formula I.
  • the RNAi sense modified oligonucleotide comprises at least two, at least three, at least four, at least five, or at least six modified intemucleoside linking groups having Formula I.
  • the RNAi antisense modified oligonucleotide comprises exactly one modified intemucleoside linking group having Formula I. In certain embodiments, the RNAi antisense modified oligonucleotide comprises exactly two modified intemucleoside linking groups having Formula I. In certain embodiments, the RNAi antisense modified oligonucleotide comprises exactly three modified intemucleoside linking groups having Formula I.
  • the RNAi antisense modified oligonucleotide comprises exactly four modified intemucleoside linking groups having Formula I.
  • the RNAi sense modified oligonucleotide comprises exactly one modified intemucleoside linking group having Formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly two modified intemucleoside linking groups having Formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly three modified intemucleoside linking groups having Formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly four modified intemucleoside linking groups having Formula I. In certain embodiments, the RNAi sense modified oligonucleotide comprises exactly five modified intemucleoside linking groups having Formula I.
  • At least one of the five 3’ -most intemucleoside linking groups of the RNAi antisense modified oligonucleotide is a modified intemucleoside linking group having Formula I. In certain embodiments, at least two of the five 3’ -most intemucleoside linking groups of the RNAi antisense modified oligonucleotide are modified intemucleoside linking groups having Formula I.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of modified oligonucleotides.
  • the above modifications are incorporated into a modified oligonucleotide.
  • modified oligonucleotides are characterized by their modifications, motifs, and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each intemucleoside linkage of a modified oligonucleotide may be modified or unmodified and may or may not follow the modification pattern of the sugar moieties.
  • modified oligonucleotides may comprise one or more modified nucleobase independent of the pattern of the sugar modifications.
  • a modified oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a region of nucleosides having specified sugar modifications), in such circumstances it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied.
  • a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions or segments, A, B, and C, wherein region or segment A consists of 2-6 linked nucleosides having a specified sugar moiety, region or segment B consists of 6-10 linked nucleosides having a specified sugar moiety, and region or segment C consists of 2-6 linked nucleosides having a specified sugar moiety.
  • Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide is 22, which exceeds the upper limit of 20 for the overall length of the modified oligonucleotide.
  • all modifications are independent of nucleobase sequence except that the modified nucleobase 5-methylcytosine is necessarily a “C” in an oligonucleotide sequence.
  • nucleobase T is replaced with the nucleobase U.
  • oligonucleotides consist of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number nucleosides in the range.
  • X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • oligonucleotides consist of 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17,
  • oligonucleotides have a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • a region of an oligonucleotide has a nucleobase sequence that is complementary to a second oligonucleotide or an identified reference nucleic acid, such as a target nucleic acid.
  • the nucleobase sequence of a region or entire length of an oligonucleotide is at least 70%, at least 80%, at least 90%, at least 95%, or 100% complementary to the second oligonucleotide or nucleic acid, such as a target nucleic acid.
  • antisense agents, oligomeric compounds, and modified oligonucleotides described herein comprise or consist of a modified oligonucleotide that optionally comprises a conjugate group.
  • Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position.
  • conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide.
  • conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups.
  • conjugate moieties or terminal groups are attached at the 3 ’ and/or 5’ -end of oligonucleotides.
  • conjugate moieties are attached at the 3’ -end of oligonucleotides. In certain embodiments, conjugate moieties are attached near the 3’ -end of oligonucleotides. In certain embodiments, conjugate moieties (or terminal groups) are attached at the 5’ -end of oligonucleotides. In certain embodiments, conjugate moieties are attached near the 5’ -end of oligonucleotides.
  • At least one intemucleoside linkage has formula I: wherein R comprises a conjugate group. In certain embodiments, R is Ci 6.
  • modified oligonucleotides comprise one or more conjugate moieties or conjugate groups.
  • conjugate groups modify one or more properties of the molecule, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • conjugate moieties impart a new property on the molecule, e.g., fluorophores or reporter groups that enable detection of the molecule.
  • conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem.
  • cholesterol moiety Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556
  • cholic acid Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060
  • a thioether e.g.,
  • a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO ./., 1991, 10, 1111-1118; Kabanov et al., FEBSLett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J Pharmacol. Exp.
  • Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
  • intercalators include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, bio
  • a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofcn carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • an active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofcn car
  • conjugate groups comprise a conjugate linker that attaches a conjugate moiety to the remainder of the modified oligonucleotide.
  • a conjugate linker is a single chemical bond (i.e. conjugate moiety is attached to the remainder of the modified oligonucleotide via a conjugate linker through a single bond).
  • the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units such as ethylene glycol, nucleosides, or amino acid units.
  • a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphoms moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
  • conjugate linkers are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to oligomeric compounds, such as the oligonucleotides provided herein.
  • a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on an oligomeric compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include but are not limited to electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
  • conjugate linkers include but are not limited to pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxy late (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
  • ADO 8-amino-3,6-dioxaoctanoic acid
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxy late
  • AHEX or AHA 6-aminohexanoic acid
  • conjugate linkers include but are not limited to substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 2 -C 10 alkenyl or substituted or unsubstituted C 2 -C 10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • conjugate linkers comprise 1-10 linker-nucleosides.
  • such linker-nucleosides are modified nucleosides.
  • such linker-nucleosides comprise a modified sugar moiety.
  • linker-nucleosides are unmodified.
  • linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine.
  • a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N- benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N- isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the oligomeric compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the oligomeric compound through cleavable bonds.
  • conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
  • a conjugate group or conjugate moiety it is desirable for a conjugate group or conjugate moiety to be cleaved from the remainder of the oligonucleotide.
  • oligomeric compounds including oligomeric compounds that are antisense agents or portions thereof
  • modified oligonucleotides comprising a particular conjugate moiety are better taken up by a particular cell type, but once the compound has been taken up, it is desirable that the conjugate group be cleaved to release an unconjugated oligonucleotide.
  • certain conjugate moieties may comprise one or more cleavable moieties, typically within the conjugate linker.
  • a cleavable moiety is a cleavable bond.
  • a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
  • a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate or phosphodiester linkage between an oligonucleotide and a conjugate moiety or conjugate group.
  • a cleavable moiety comprises or consists of one or more linker-nucleosides.
  • one or more linker-nucleosides are linked to one another and/or to the remainder of the oligomeric compound through cleavable bonds.
  • such cleavable bonds are unmodified phosphodiester bonds.
  • a cleavable moiety is a nucleoside comprising a 2'-deoxyfuranosyl that is attached to either the 3' or 5'-terminal nucleoside of an oligonucleotide by a phosphodiester intemucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphodiester or phosphorothioate linkage.
  • the cleavable moiety is a nucleoside comprising a 2 ' - ⁇ -D-dcoxyribosyl sugar moiety.
  • the cleavable moiety is 2’-deoxyadenosine.
  • a conjugate group comprises a cell-targeting conjugate moiety.
  • a conjugate group has the general formula:
  • n is from 1 to about 3, m is 0 when n is 1. m is 1 when n is 2 or greater, j is 1 or 0, and k is 1 or
  • n is 1, j is 1 and k is 0. In certain embodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1, j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0. In certain embodiments, n is 2, j is O andk is l. In certain embodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1. In certain embodiments, n is 3, j is 1 and k is 1.
  • conjugate groups comprise cell-targeting moieties that have at least one tethered ligand.
  • cell-targeting moieties comprise two tethered ligands covalently attached to a branching group.
  • cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.
  • the cell-targeting moiety comprises a branching group comprising one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups.
  • the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups.
  • the branched aliphatic group comprises groups selected from alkyl, amino, oxo, amide and ether groups.
  • the branched aliphatic group comprises groups selected from alkyl, amino and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system.
  • each tether of a cell-targeting moiety comprises one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amino, oxo, amide, phosphodiester, and polyethylene glycol, in any combination.
  • each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol, in any combination.
  • each tether is a linear aliphatic group comprising one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide, in any combination.
  • each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, amino, oxo, and amid, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, amino, and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group.
  • each tether comprises a chain from about 6 to about 20 atoms in length. In certain embodiments, each tether comprises a chain from about 10 to about 18 atoms in length. In certain embodiments, each tether comprises about 10 atoms in chain length.
  • each ligand of a cell-targeting moiety has an affinity for at least one type of receptor on a target cell. In certain embodiments, each ligand has an affinity for at least one type of receptor on the surface of a mammalian lung cell.
  • the cell-targeting moiety has affinity for the Asialoglycoprotein receptor (ASGPR).
  • each ligand of a cell-targeting moiety is a carbohydrate, carbohydrate derivative, modified carbohydrate, polysaccharide, modified polysaccharide, or polysaccharide derivative.
  • the conjugate group comprises a carbohydrate cluster (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al., “Design and Synthesis of Novel Y- Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” ./. Med. Chem. 2004, 47, 5798-5808, which are incorporated herein by reference in their entirety).
  • each ligand is an amino sugar or a thio sugar.
  • amino sugars may be selected from any number of compounds known in the art, such as sialic acid, ⁇ -D- galactosamine, ⁇ -muramic acid, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl- D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-glycolyl- ⁇ -neuraminic acid.
  • thio sugars may be selected from 5 -Thio- ⁇ -D-glucopyranose, methyl 2,3,4-tri-O-acetyl-l-thio-6-O- trityl- ⁇ -D-glucopyranoside, 4-thio- ⁇ -D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-l,5-dithio- ⁇ -D-g/nco- heptopyranoside.
  • oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) or modified oligonucleotides described herein comprise a conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., IntJ Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessenet al., JMed Chem, 1995, 38, 1538- 1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487
  • the conjugate group comprises N-acetylgalactosamine (GalNAc).
  • the conjugate group is attached to the first modified oligonucleotide at the 5’ -end of the first modified oligonucleotide. In certain embodiments, the conjugate group is attached to the first modified oligonucleotide at the 3’ -end of the modified oligonucleotide.
  • the conjugate group comprises a cell-targeting moiety having an affinity for transferrin receptor (TfR), also known as TfRl and CD71.
  • TfR transferrin receptor
  • the conjugate group comprises an anti-TfRl antibody or fragment thereof.
  • the conjugate group comprises a peptide capable of binding TfRl.
  • the conjugate group comprises an aptamer capable of binding TfRl.
  • compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising one or more oligomeric compounds (including oligomeric compounds that are antisense agents or portions thereof) or a salt thereof.
  • the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises a sterile saline soluhon and one or more oligomeric compound.
  • such pharmaceutical composition consists of a sterile saline solution and one or more oligomeric compound.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composihon comprises one or more oligomeric compound and sterile water.
  • a pharmaceutical composihon consists of one oligomeric compound and sterile water.
  • the sterile water is pharmaceutical grade water.
  • a pharmaceutical composition comprises or consists of one or more oligomeric compound and phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • a pharmaceutical composihon consists of one or more oligomeric compound and sterile PBS.
  • the sterile PBS is pharmaceutical grade PBS.
  • an oligomeric compound described herein complementary to a target nucleic acid can be utilized in pharmaceutical compositions by combining the oligomeric compound with a suitable pharmaceutically acceptable diluent or carrier and/or additional components such that the pharmaceutical composition is suitable for injection.
  • a pharmaceutically acceptable diluent is phosphate buffered saline.
  • employed in the methods described herein is a pharmaceutical composition comprising an oligomeric compound complementary to a target nucleic acid and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is phosphate buffered saline.
  • the oligomeric compound comprises or consists of a modified oligonucleotide provided herein.
  • compositions comprising oligomeric compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the oligomeric compound comprises or consists of a modified oligonucleotide. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • antisense agents, oligomeric compounds, or modified oligonucleotides described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid.
  • the target nucleic acid is an endogenous RNA molecule.
  • the target nucleic acid encodes a protein.
  • the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions.
  • the target RNA is an mRNA.
  • the target nucleic acid is a pre-mRNA.
  • a pre-mRNA and corresponding mRNA are both target nucleic acids of a single compound.
  • the target region is entirely within an intron of a target pre-mRNA.
  • the target region spans an intron/exon junction.
  • the target region is at least 50% within an intron.
  • the target nucleic acid is a microRNA.
  • the target region is in the 5’ UTR of a gene.
  • the target region is within a translation suppression element region of a target nucleic acid.
  • Certain compounds described herein e.g., antisense agents, oligomeric compounds, and modified oligonucleotides
  • Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds.
  • Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms. All tautomeric forms of the compounds provided herein are included unless otherwise indicated.
  • the compounds described herein include variations in which one or more atoms are replaced with a non- radioactive isotope or radioactive isotope of the indicated element.
  • compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the 1 H hydrogen atoms.
  • Isotopic substitutions encompassed by the compounds herein include but are not limited to: 2 H or 3 H in place of 1 H, 13 C or 14 C in place of 12 C, 15 N in place of 14 N, 17 O or 18 O in place of 16 O, and 33 S, 34 S, 35 S, or 36 S in place of 32 S.
  • non-radioactive isotopic substitutions may impart new properties on the oligomeric compound that are beneficial for use as a therapeutic or research tool.
  • radioactive isotopic substitutions may make the compound suitable for research or diagnostic purposes such as imaging.
  • oxidizing solutions each comprising sulfonyl oxidizing agent methanesulfonyl azide (mesyl azide, MsN 3 ) were prepared.
  • One oxidizing solution contained no stabilizing agent (control solution), and four oxidizing solutions each included a stabilizing agent.
  • the stabilizing agents tested were triphenyl phosphate (TPP) and diphenyl sulfone (DPS):
  • NaN 3 (5.4 g, 83 mmol, 1 eq) was suspended in dry acetonitrile (MeCN, 80 mL), and was cooled to 0 °C while stirring under nitrogen.
  • Mesyl chloride (10 g, 6.75 ml, 88.3 mmol, 1.05 eq.) was added dropwise, and the reaction was allowed to warm to room temperature over the course of 3 hours. The reaction was filtered to remove insoluble salts, resulting in the desired 1.0 M MsN 3 solution in MeCN.
  • Oxidizing solutions 1-3 described above were tested by Dekra (dekra.us/process-safety) using their standard protocol Briefly, solvent was removed from each oxidizing solution, and the corresponding residues were evaluated by differential scanning calorimetry (D SC) to determine their heats of composition. This test determines the onset temperature of any energetic events and the total energy associated with those events. Each event is described in a separate row in Table 2 below and labeled for whether there was a positive (endotherm) or negative (exotherm) change in enthalpy of the residue as the residue is heated.
  • An energy of decomposition greater than 300 J.g -1 indicates a highly energetic material, and an energy of decomposition greater than 500 J.g -1 suggests the material may have explosive properties.
  • residues containing TPP have lower combustion energies than the residue from the control solution without any solid stabilizing agent (oxidizing solution 1).
  • Example 3 Synthesis of modified oligonucleotides containing mesyl phosphoramidate internucleoside linkages via oxidative mesylation using mesyl azide solutions
  • the following figure shows a general, high-level scheme of oligonucleotide synthesis using mesyl azide as an oxidizing agent.
  • An oligonucleotide intermediate displayed as a black and white ribbon, is attached onto a solid support (shown as a circle).
  • a phosphoramidite monomer is incorporated onto the oligonucleotide using standard techniques.
  • B(pg) in the figure below represents a variable nucleobase with generic protecting group(s).
  • Modified oligonucleotides comprisng mesyl phosphoramidate intemucleoside linkages were prepared using the oxidizing solutions described in Example 1.
  • the oligonucleotide intermediate, Compound A was synthesized using standard techniques.
  • Compound A is a modified oligonucleotide intermediate 13 nucleosides in length having nucleobase sequence (from 5' to 3'): TGGTTATGACTCA (SEQ ID NO: 1).
  • the sugar motif of Compound A is (from 5' to 3'): dddddddeeeee; wherein each “d” represents a 2 ‘ - ⁇ -D-dcoxyribosyl sugar moiety, and each “e” represents a 2’-MOE sugar moiety.
  • the intemucleoside linkage motif of Compound A is (from 5’ to 3’): sssssssssss, wherein each “s” represents a phosphorothioate intemucleoside linkage.
  • Each cytosine residue is a 5-methylcytosine.
  • the 5’-OH of Compound A is capped with a dimethoxytrityl (DMT) protecting group.
  • DMT dimethoxytrityl
  • the linked nucleosides of Compound A are attached to a solid support. Two 2 ‘ - ⁇ -D-dcoxyribosyl thymidine nucleosides were joined to Compound A via mesyl phosphoramidate intemucleoside linkages.
  • Modified oligonucleotides were synthesized on an AKTA Oligopilot 10 (35 ⁇ mol scale).
  • Deoxy T phosphoramidite was dissolved in 1 : 1 MeCN/toluene (v/v) and was dried over molecular sieves. The DMT protecting group was removed from the modified oligonucleotide intermediate using 15% DCA in toluene.
  • Deoxy T phosphoramidite was coupled using 3 equivalents of amidite and 10 equivalents of activator (1 M 4,5-dicyanoimidazole and 0.1 M Y-mcthylimidazolc in acetonitrile) relative to amidite, and the coupling solution was allowed to recycle for 6 minutes.
  • the oxidizing solution comprising MsN 3 was added (25 equiv) and was allowed to recycle for 25 minutes followed by an MeCN wash. After washing, the reaction mixture was treated with 20% acetic anhydride in MeCN (Cap A) and N- methylimidazole in MeCN/pyridine (2:5:3 v/v/v, Cap B) to cap any coupling failures. The cycle was repeated to incorporate the second mesyl-linked thymidine nucleoside.
  • the modified oligonucleotide intermediate, Compound B was cleaved from solid support and deprotected using standard techniques to yield the final modified oligonucleotide.
  • Compound B is a modified oligonucleotide 15 nucleosides in length having sequence (from 5’ to 3’): TTTGGTTATGACTCA (SEQ ID NO: 2).
  • the sugar motif of Compound B is (from 5’ to 3’): ddddddddddeeeee; wherein each “d” represents a 2 ' - ⁇ -D-dcoxyribosyl sugar moiety, and each “e” represents a 2’-MOE sugar moiety.
  • the intemucleoside linkage motif of Compound B is (from 5’ to 3’): zzsssssssss, wherein each “s” represents a phosphorothioate intemucleoside linkage, and each “z” represents a mesyl phosphoramidate intemucleoside linkage.
  • Each cytosine residue is a 5-methylcytosine.
  • Oxidizing solutions 1-5 were each used to synthesize Compound B separately, resulting in products analogously labeled Compounds Bl, B2, B3, B4, and B5.
  • Compounds B1-B5 were analyzed by UV chromatography and liquid chromatography-mass spectrometry.
  • oxidizing solution 2 TPP in 1:1 toluene to MeCN
  • the stabilizing agents appeared to have no deleterious effect on coupling and may reduce hazardous risk of working with mesyl azide.
  • Example 4 Preparation of methanesulfonyl azide solution in acetonitrile with sulfolane as a stabilizing agent
  • oxidizing solution comprising sulfonyl oxidizing agent MsN 3 in acetonitrile, including sulfolane as a stabilizing agent was prepared.
  • the structure of sulfolane is shown below:
  • the solution was prepared using two methods, as described below.
  • the reaction was confirmed complete based on the absence of the chemical shift for the mesyl chloride (expected: 3.8 ppm) by 1 H NMR.
  • the concentration of MsN 3 was determined to be 2.082 M by quantitative NMR using ethylene carbonate as the analytical standard.
  • the reaction was then filtered through a bottle-top filter into a tared polycoated glass bottle with Teflon coated- magnetic stir bar.
  • the filter cake was rinsed with a small volume of MeCN ( ⁇ 10 mL).
  • the bottle was then charged with melted sulfolane (621.28g, 5.17 mol).
  • the mixture was stirred, and the solution mass and density were determined and used to dilute the solution with MeCN to a total volume of 1034 mL.
  • the reaction was confirmed complete based on the absence of the chemical shift for the Mesyl chloride (expected: 3.8 ppm) by 1 H NMR.
  • the concentration of MsN 3 was determined to be 0.943 M in the reaction mixture by quantitative NMR using ethylene carbonate as the analytical standard.
  • reaction was then filtered through a bottle-top filter into a tared polycoated glass bottle and stored without further dilution.
  • Example 5 Synthesis of modified oligonucleotides containing mesyl phosphoramidate internucleoside linkages via oxidative mesylation using mesyl azide solutions
  • Modified oligonucleotides comprising mesyl phosphoramidate intemucleoside linkages were prepared using a solution of MSN 3 in acetonitrile with sulfolane.
  • Modified oligonucleotides were synthesized on an AKTA Oligopilot 10 (40 ⁇ mol scale) using polystyrene based NittoPhase HL UnyLinker support (405 ⁇ mol /g). Fully protected nucleoside phosphoramidites were incorporated using standard solid-phase modified oligonucleotide synthesis conditions, described herein above in Example 3. DNA amidites were dissolved at 0.1 M in 1:1 MeCN/toluene and incorporated using 6 min recycling times. 1 M 4,5-dicyanoimidazole with 0.1 M N-methylimidazole in MeCN was used as an activator. DMT protecting group were removed using 15% dichloroacetic acid in toluene. 20% acetic anhydride in MeCN and N-methylimidazole/pyridine/MeCN (20:30:50) was used for capping coupling failures.
  • Oxidations of the P(III) species were performed as follows: 0.05 M iodine in pyridine/FLO (9:1) for phosphodiester linkages; or 0.1 M xanthane hydride in 1:1 pyridine:MeCN for phosphorothioate linkages.
  • the modified oligonucleotide intermediate was treated with 0.65 M MSN in 1:1 MeCN:sulfolane or 0.65 M MsN in MeCN and allowed to recycle for 25 minutes.
  • the cyanoethyl protecting groups were removed using 20% diethylamine in toluene, and the remaining protecting groups were cleaved by suspending the solid support in aqueous concentrated ammonia and heating at 55 °C for 14 h.
  • the support was removed by filtration and the crude mixture was purified by HPLC using a combined purification, detritylation, desalt method.
  • the basic RSR (Reverse phase, SAX, Reverse phase) method the sample was loaded onto the RP column (DuPont XT30) in H 2 O. A failure elution was then performed on the RP column with 1:1 (A: 80% MeOH/water, B : 2.5 M NaCl, 50 mM NaOH).
  • DMT cleavage was then performed on the RP column with 6% DCA, followed by a water wash.
  • the detritylated compound was loaded onto the SAX column with 80% MeOH.
  • the RP column was equilibrated with 50 mM NaOH.
  • a SAX gradient was then performed from 0 to 50% with A and B buffers (A: 50 mM NaOH, B: 50 mM NaOH, 2.5 M NaCl).
  • a and B buffers A: 50 mM NaOH, B: 50 mM NaOH, 2.5 M NaCl.
  • Modified oligonucleotide Compound 1633475 was synthesized using standard techniques described herein above, with and without sulfolane, and each lot was analysed for lot purity.
  • Compound 1633475 is a modified oligonucleotide intermediate 16 nucleosides in length having nucleobase sequence (from 5' to 3'): GCATGTTCTCACATTA (SEQ ID NO: 3).
  • the sugar motif of Compound 1633475 is (from 5' to 3'): kkkddddddddddkkk; wherein each “d” represents a 2 ' - ⁇ -D-dcoxyribosyl sugar moiety, and each “k” represents a cEt sugar moiety.
  • the intemucleoside linkage motif of Compound 1633475 is (from 5’ to 3’): ssszzzzsssssss, wherein each “s” represents a phosphorothioate intemucleoside linkage, and each “z” represents a mesyl phosphoramidate intemucleoside linkage. Each cytosine residue is a 5-methylcytosine.
  • Compound 1633475 further contains a 3THAGNhp moiety conjugated to the 3'-terminal oxygen of the modified oligonucleotide via a phosphodiester bond as shown below:
  • 3THAGNhp A sample of each modified oligonucleotide lot was made in 0.01% triethylamine in H 2 O at an approximate concentration of 1 mg/mL. The samples were analyzed by Ion Pair HPLC/mass spectrometry (IP-HPLC/MS) on an Agilent 1200 series equipped with a binary pump, online degasser, heated column chamber, autosampler, and multiple wavelength UV detector, interfaced to an electrospray mass spectrometer. Analysis was performed using a Waters (Milford, MA, USA) XBridgeTM HPLC column (18C, 3.5 pm, 2.1 x 150 mm, Waters P/N 186003023).
  • IP-HPLC/mass spectrometry IP-HPLC/mass spectrometry
  • the UV absorbance of column eluate was measured at 260 nm, using a reference wavelength of 400 nm.
  • Column eluate was introduced directly into the ESI- MS.
  • the ESI source was operated in negative mode, with a scanning mass signal (m z) range of (full length product mass)/4 ⁇ 150.0.
  • the full-length product (main UV peak), early eluting impurity, and late eluting impurity UV peaks at 260 nm were identified and integrated in OpenLab ChemStation version C.01.09.
  • the area of the main UV peak was normalized to the sum area of all peaks at 260 nm and is presented in the tables below as UV Purity (%).
  • the full-length product m/z and all impurity m/z were identified within the main UV peak.
  • the ion chromatogram for each component mass signal was extracted and integrated.
  • the area of the full-length product signal was normalized to the sum of the component signals and is presented below as MS purity (%).
  • MS purity %.
  • Example 7 Impact sensitivity analysis of mesyl azide in the presence of sulfolane

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Abstract

La présente invention concerne des procédés de synthèse d'oligonucléotides modifiés et de composés oligomères (comprenant des composés oligomères qui sont des agents antisens ou des parties de ceux-ci) comprenant un oligonucléotide modifié ayant au moins un groupe de liaison internucléoside modifié. Dans certains modes de réalisation, la présente invention concerne des formulations stabilisées de certains azides de sulfonyle destinées à être utilisées dans la synthèse d'oligonucléotides comprenant une ou plusieurs liaisons de phosphoramidate de sulfonyle. Certains modes de réalisation concernent des compositions stabilisées de réactifs à haute énergie qui peuvent être utilisées dans la synthèse d'oligonucléotides modifiés, permettant un processus sécurisé de préparation à l'échelle industrielle.
EP22834149.1A 2021-06-30 2022-06-29 Procédé de synthèse de composés oligomères modifiés par liaison Pending EP4363572A1 (fr)

Applications Claiming Priority (2)

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US202163217137P 2021-06-30 2021-06-30
PCT/US2022/035539 WO2023278589A1 (fr) 2021-06-30 2022-06-29 Procédé de synthèse de composés oligomères modifiés par liaison

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EP4363572A1 true EP4363572A1 (fr) 2024-05-08

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EP (1) EP4363572A1 (fr)
KR (1) KR20240027745A (fr)
CN (1) CN117480254A (fr)
WO (1) WO2023278589A1 (fr)

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JP2022544587A (ja) * 2019-08-15 2022-10-19 アイオーニス ファーマシューティカルズ, インコーポレーテッド 結合修飾オリゴマー化合物及びその使用

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WO2023278589A1 (fr) 2023-01-05
CN117480254A (zh) 2024-01-30

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